Fate Tracing Reveals the Pericyte and Not Epithelial Origin of Myofibroblasts in Kidney Fibrosis

Renal Division, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
American Journal Of Pathology (Impact Factor: 4.59). 12/2009; 176(1):85-97. DOI: 10.2353/ajpath.2010.090517
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Understanding the origin of myofibroblasts in kidney is of great interest because these cells are responsible for scar formation in fibrotic kidney disease. Recent studies suggest epithelial cells are an important source of myofibroblasts through a process described as the epithelial-to-mesenchymal transition; however, confirmatory studies in vivo are lacking. To quantitatively assess the contribution of renal epithelial cells to myofibroblasts, we used Cre/Lox techniques to genetically label and fate map renal epithelia in models of kidney fibrosis. Genetically labeled primary proximal epithelial cells cultured in vitro from these mice readily induce markers of myofibroblasts after transforming growth factor beta(1) treatment. However, using either red fluorescent protein or beta-galactosidase as fate markers, we found no evidence that epithelial cells migrate outside of the tubular basement membrane and differentiate into interstitial myofibroblasts in vivo. Thus, although renal epithelial cells can acquire mesenchymal markers in vitro, they do not directly contribute to interstitial myofibroblast cells in vivo. Lineage analysis shows that during nephrogenesis, FoxD1-positive((+)) mesenchymal cells give rise to adult CD73(+), platelet derived growth factor receptor beta(+), smooth muscle actin-negative interstitial pericytes, and these FoxD1-derivative interstitial cells expand and differentiate into smooth muscle actin(+) myofibroblasts during fibrosis, accounting for a large majority of myofibroblasts. These data indicate that therapeutic strategies directly targeting pericyte differentiation in vivo may productively impact fibrotic kidney disease.

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Available from: Ben Humphreys, May 16, 2014
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    • "Loss of stromal miRNAs results in a multifaceted renal phenotype The FoxD1-positive cortical renal stroma in the developing kidney marks a stromal progenitor population that gives rise to diverse cell lineages, including the cortical and medullary interstitial cells, glomerular mesangial cells, and pericytes (Kobayashi et al. 2014). To delineate the role of stromal miRNAs in renal development, we ablated Dicer, an endoribonuclease required for miRNA biogenesis , in the renal stroma and its derivatives by generating FoxD1 GC ;Dicer fl/fl transgenic mice (Harfe et al. 2005; Humphreys et al. 2010). To evaluate the excision of Dicer exon 24 from the Dicer fl allele by the FoxD1 GC allele, we performed quantitative real-time PCR and observed a significant decrease in exon 24 (***P < 0.001) but not exons 21 and 23 transcripts in our mutant model (Fig. 1A). "
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    ABSTRACT: MicroRNAs are small noncoding RNAs that post-transcriptionally regulate mRNA levels. While previous studies have demonstrated that miRNAs are indispensable in the nephron progenitor and ureteric bud lineage, little is understood about stromal miRNAs during kidney development. The renal stroma (marked by expression of FoxD1) gives rise to the renal interstitium, a subset of peritubular capillaries, and multiple supportive vascular cell types including pericytes and the glomerular mesangium. In this study, we generated FoxD1(GC);Dicer(fl/fl) transgenic mice that lack miRNA biogenesis in the FoxD1 lineage. Loss of Dicer activity resulted in multifaceted renal anomalies including perturbed nephrogenesis, expansion of nephron progenitors, decreased renin-expressing cells, fewer smooth muscle afferent arterioles, and progressive mesangial cell loss in mature glomeruli. Although the initial lineage specification of FoxD1(+) stroma was not perturbed, both the glomerular mesangium and renal interstitium exhibited ectopic apoptosis, which was associated with increased expression of Bcl2l11 (Bim) and p53 effector genes (Bax, Trp53inp1, Jun, Cdkn1a, Mmp2, and Arid3a). Using a combination of high-throughput miRNA profiling of the FoxD1(+)-derived cells and mRNA profiling of differentially expressed transcripts in FoxD1(GC);Dicer(fl/fl) kidneys, at least 72 miRNA:mRNA target interactions were identified to be suppressive of the apoptotic program. Together, the results support an indispensable role for stromal miRNAs in the regulation of apoptosis during kidney development.
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    • "To preserve β-galactosidase activity, liver tissue was fixed in PLP (4% paraformaldehyde (PFA), 75 mM l-lysine, 10 mM sodium periodate) for 2 hours at 4°C, cryopreserved in 18% sucrose, frozen in optimum cutting temperature compound, and 5-µm cryosections were prepared for X-gal labeling or immunofluorescence (IF). LacZ activity was measured using a standard 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal) staining protocol [46] for 16 hours at 37°C. After washing, sections were post-fixed in 1% PFA for 5 minutes and immunolabeled for additional histologic analysis (see below). "
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    PLoS ONE 10/2014; 9(10):e108505. DOI:10.1371/journal.pone.0108505 · 3.23 Impact Factor
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    • "No therapy has been established to prevent renal fibrosis. Since many different kinds of cell, such as immune cells, vascular endothelial cells, tubular epithelial cells and fibroblasts, have been considered to contribute to the development of renal fibrosis, the investigation of therapeutic approaches, including the exploration of target genes, for the inhibition of renal fibrosis in vivo is crucially important to improve the prognosis of all chronic kidney diseases56789101112. "
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    ABSTRACT: Renal fibrosis is the final common pathway leading to decreased renal function. No therapy has been established to prevent it. In order to establish a therapeutic approach and target molecule for renal fibrosis, we investigated the effects of Smad4 knockdown by siRNAs on renal fibrosis in vivo. Renal fibrosis mice were produced by single intraperitoneal injection of folic acid. siRNAs targeted to Smad4 (Smad4-siRNAs) (5 nmol) were injected into each mouse by systemic tail vein injection three times per week. Non-targeted siRNAs (control-siRNAs) were injected in the same way for a control group. The siRNAs were delivered to the interstitial fibrous area and tubules. Smad4-siRNAs significantly knocked down Smad4 expression and inhibited renal fibrosis. They also inhibited α-SMA-positive myofibroblasts. Control-siRNAs did not show these effects. The results of this study suggest that Smad4 knockdown is one of the crucial therapeutic options for the prevention of renal fibrosis in vivo.
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